Lipids are the building blocks of cell membranes. Scientists are continually finding new ways to use lipid-based constructs in applications, such as but not limited to drug delivery and gene transfection. In scientific studies lipids are used as materials for constructing model lipid bilayers (planar bilayers, giant unilamellar vesicles, large and small unilamellar vesicles) and other lipid-based constructs such as but not limited to nanodiscs, and nanocarriers. Other lipid-like molecules, membrane components, and even synthetic molecules are used in the creation of vesicles and self-assembled aggregates in solution. Such molecules may be charged or uncharged and include lipids as well as cholesterol, signaling lipids, lipid A, cardiolipin, proteins, surfactants, cell-derived lipids and molecules, and amphiphilic polymers and copolymers among other species. Often, lipids or similar amphiphiles are dissolved in an aqueous medium, such as but not limited to buffered water, and are further processed to produce unilamellar vesicles (UV) or liposomes. When the lipids or amphiphiles are initially dissolved, their amphiphilic structure drives self-assembly into sheets such that the hydrophobic lipid tails are not exposed to the surrounding water medium. Most commonly, the resulting structures are multilamellar vesicles (MLVs), or layered onion-like spheres (vesicles/liposomes) where each layer is comprised of the self-assembled lipids or amphiphiles. MLVs may also be formed by injecting organic solvent containing amphiphiles into aqueous solution. For many applications, MLVs are further processed to produce smaller unilamellar vesicles (SUVs) or liposomes by extrusion through a membrane with pores of specific diameter (typically 100-500 nm diameter). Extrusion is the most common method of processing and preparing liposomes.
In addition to extrusion, there are other established methods for sizing liposomes including emulsification, homogenization, microfluidization, organic-solvent injection, reverse phase evaporation, or sonication via a tip or bath style sonicator that delivers high energy to break up the MLVs into smaller UVs. Additionally, other novel techniques for liposome formation continues to emerge. The present invention specifically lies in the field of extrusion. In some cases, the lipids or amphiphiles may be dissolved in oil, placed in an oil-water emulsion, or placed in a double-emulsion or foam. The resulting colloidal structure may still be extruded to adjust their size and structure. Extrusion is also applicable for the sizing and processing of colloidal structures formed from polymers, copolymers, cells, cellular lipid extracts, cellular exosomes and liposomes and membrane “blebs”, even cellular plasma membrane vesicles. Applications of extruded liposomes range from basic fundamental physical, chemical, and biological research to medical use as therapeutics, adjuvants, or delivery vehicles for drugs and small molecules, and also to cosmetic, agricultural, nutrition and nutraceutical, and food and beverage and dietary supplement industries.
There are existing devices that are used for extrusion, and these devices can be classified into categories: those intended for extrusion of small volumes (<1-10 mL) and those intended for extrusion of large volumes (from 1 mL up to 1 L, 10 L, or more). The present invention described herein can be used to extrude small or large volumes, but the present invention is specifically targeted towards at improving large-volume extrusion. Existing large-volume extrusion options are all similar, relying on high gas-pressure to drive liposome suspensions through a membrane with pores of defined and desired diameter. Examples of such currently available high-pressure extrusion systems include Lipex LiposoFast and Maximator systems. A deficiency with existing technology such as the aforementioned is the inability to precisely control the number of passes for a solution. Some systems only permit one extrusion per solution pass then require steps to initiate a second cycle or pass of the solution. As it is commonly required for many cycles or passes to achieve the required extrusion this can be time consuming. Additionally, existing technology a mixture of unextruded and extruded solution can be returned to the source container which leads to accuracy when determining a correct quantity of cycles and/or passes to extrude a solution.
Unlike existing devices and systems used for extrusion of small and large volumes of solution, the present invention disclosed herein can be used without having to “pour” or flow the suspension into any reusable part of the device that must be cleaned between uses. Avoidance of suspension contact with reusable parts is essential given that reusable parts are exposed to solutions and must thus be cleaned thoroughly, and possibly sterilized, i.e. for pharmaceutical or medical preparation or use, between production runs of different batches or lots of solutions. The need to clean, assemble, extrude, and clean again leads to a significant amount of lost time and cost in every single batch of extrusion. Utilization of these types of extruders leads to significant risk of contamination or carryover from sample to sample, despite the required rigorous cleaning procedures. Cleaning is particularly difficult if not impossible, often requiring harsh organic and environmentally unfriendly solvents like chloroform, ethanol, methanol, acetonitrile, or other solvents. Cleaning proves extremely difficult if cholesterol, proteins, membrane receptors and proteins, or other drugs, biomolecules, nanoparticles, and small molecules containing hydrophobic regions are used. In some cases, cleaning requires specialized harsh detergents and treatment, all of which increases cost and introduces significant risk of contamination. It is known in the art that the inherent risks associated with the need for cleaning which, even when done properly and according to manufacturer's recommendations, often fail to completely remove all material resulting in trace contaminants left behind. For applications in medical and pharmaceutical, food, cosmetic, and other industries, contamination due to improper cleaning and assembly could cause, infection physical harm to patients and even death, unwanted side effects, unintended consequences, blockage of capillaries and blood flow, and otherwise detrimental outcomes.
Accordingly, there is a need for continuous extrusion system that has no upper limit on the total volume that can be processed, controls the number of cycles and/or passes of a solution and further uses specially designed kits wherein the extrudant solution preferably comes into contact with only single-use, disposable components.